SUMMARY ABSTRACT: CRISPR-Modified Cardiac Xenograft Transplantation The past three years have witnessed two major breakthroughs in heart xenotransplantation. First, the previously intractable barrier of delayed xenograft rejection (DXR) has been overcome in the pig-to-baboon heart xeno model. In this model, DXR is manifest primarily as consumptive coagulopathy (CC) in the recipient and thrombotic microangiopathy (TM) in the graft. Working in the heterotopic model in baboons, our `Mohiuddin/NIH' group used genetically modified GTKO.hCPRP.hTBM hearts and a treatment regimen including ?induction? T and B depletion, MMF, and steroids ? all clinically used drugs ? with an experimental monoclonal antibody directed against CD40. DXR was prevented for as long as ?CD40 treatment was continued, and in one instance xenograft survival was extended beyond two years. The second major advance came from the Munich group, who reported consistent survival of orthotopic pig heart xenograft recipients beyond 180 days using our immunomodulatory regimen and GTKO.hCPRP.hTBM hearts. Accomplishing consistent survival of orthotopic heart xenografts had previously been prevented by initial xenograft dysfunction (IXD), a phenomenon refractory to concerted efforts by multiple experienced clinical transplant teams. Importantly, the Munich group found that minimizing graft ischemia was necessary and sufficient to consistently prevent IXD. Discovering that ischemia minimization was sufficient to overcome the IXD barrier is a `breakthrough' advance, even as the mechanism of improved graft protection by ischemia minimization is not yet well understood. In this Project, we hypothesize that IXD mechanisms that injure GTKO.hCPRP.hTBM hearts may be addressed by the additional xeno-focused genetic modifications expressed in one or more versions of Pig 2.0, created by our collaborators at eGenesis using innovative CRISPR-driven gene editing tools. Pig 2.0 is designed to address multiple known xeno-specific injury mechanisms through a combination of 12 xeno- targeted genetic modifications. This Project takes advantage of availability of several well-defined versions of Pig 2.0, our deep experience with multiple in vitro, ex vivo, and in vivo heart xeno models, and a robust mechanistic assay capability to determine whether Pig 2.0 is protected from heart IXD, with or without ischemia minimization (Aim 1). Specifically, we will use these unique resources to learn a) whether versions of Pig 2.0 containing a coagulation cascade regulatory gene cassette are protected from IXD; b) whether ischemia minimization is necessary and/or sufficient to prevent IXD in susceptible Pig 2.0 hearts; and c) to define a clinically acceptable immunosuppression regimen for Pig 2.0 hearts in baboons that is consistently safe and effective (Aim 2). Results from this Project will yield insights likely to catalyze progress for other cell and organ xenografts, and inform clinical heart xenotransplantation trial design.